Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure

A sodium dodecyl sulfate-gel electrophoretic procedure which allows the separation of isolated cytochrome c oxidase from different mammalian sources into 13 different polypeptides is described. Application of the silver-staining procedure results in the same protein pattern as obtained by Coomassie blue staining. From the correlation of the gel bands with 12 isolated polypeptides from which the complete amino acid sequence is known, it is concluded that mammalian cytochrome c oxidase consists of 13 different polypeptides which can all be separated by the described procedure.     

Cytochrome a-type terminal oxidase derived from Thiobacillus novellus. Molecular and enzymatic properties

Cytochrome a-type terminal oxidase was purified from Thiobacillus novellus to an electrophoretically homogeneous state and some of its properties were studied.The enzyme shows absorption peaks at 428 and 602 nm in the oxidized form, and at 442 and 602 nm in the reduced form. The CO compound of the reduced enzyme shows peaks at 431 and 599 nm. The enzyme has 1 mol of haem a and 1 g-atom of copper per 55 600 g and is composed of two kinds of subunit, of 32 000 and 23 000 daltons, respectively.The enzyme reacts rapidly with tuna, bonito and yeast cytochromes c as well as with T. novellus cytochrome c, while it reacts slowly with horse and cow cytochromes c. The reduction product of oxygen catalysed by the enzyme is water.     

Bilayer thickness and enzymatic activity in the mitochondrial cytochrome c oxidase and ATPase complex

The antigenic cross-reactivity between purified chick, eel and mouse electrolectins (endogenous β-D-galactoside specifc lectins) have been studied using a solid phase radioimmunoassay. The immune serum raised against the eel electrolectin crossreacts both with the chick and the mouse electrolectins, while the anti-chick electrolectin anti-serum recognizes only the eel but not the mouse electrolectin. These findings are analyzed in terms of the phylogenetic distance separating the species considered; they suggest that electrolectins fulfil a fundamental biological function.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH₂:cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands.

2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c₁, band 5 the Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c₁ and identified with the so-called oxidation factor, and band 7 a polypeptide associated with cytochrome b.

3. The validity of molecular weight estimates for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively.

4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide bands.     

The isolation of bovine-heart cytochrome c oxidase subunits Dependence on phospholipid and cholate content

The polypeptide chains of bovine-heart cytochrome c oxidase were preparatively isolated by a simple large-scale procedure based on gel permeation chromatography in the presence of sodium dodecyl sulphate.The resolution of the subunits as a function of the cholate and phospholipid content of the preparation was investigated.Cholate, and to a lesser extent, phospholipids interfere with the separation of the subunits; however, they do not prevent dissociation of the enzyme by SDS.Bovine-heart cytochrome c oxidase consists of six major subunits (estimated molecular weights in thousands: 40, 25, 20, 14, 12 and 10). In addition, the enzyme preparation contains at least five minor constituents, present in less than stoichiometric amounts.The first two of the three large subunits, all of which are hydrophobic, have amino-terminal N-formylmethionine. Subunit III, however, has a free methionine N-terminus.     

Topology of membrane sulfhydryl groups in the human erythrocyte Demonstration of a non-reactive population in intrinsic proteins

A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, $\text{N-}\text{ethylmaleimide}$. About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations.These ‘non-reacting’ SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labelled $\text{N-}\text{ethylmaleimide}$ or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with $\text{N-[ethyl-2-}{}^3\text{H]}\text{ethylmaleimide}$.The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive.After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards $\text{N-}\text{ethylmaleimide}$. It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.     

Formation of active 40 S ribosomal subunits in liver in the presence of aflatoxin B1

In the presence of aflatoxin B₁, 18 S RNA continues to be excised from a normal 45 S RNA, emerging into the cytoplasm in free newly synthesized 40 S subunits. The present results demonstrate that the particles so formed are indistinguishable from their control counterparts in composition and buoyant density as well as in their ability to be incorporated into polysomes. These findings suggest that 40 S subunits synthesized in the presence of aflatoxin B₁ represent active particles capable of initiating protein synthesis in rat liver cell.     

Gene 68, a new bacteriophage T4 gene which codes for the 17K prohead core protein is involved in head size determination

We have identified the gene for a major component of the prohead core of bacteriophage T4, the 17K protein. The gene, which we call gene 68, lies between genes 67 and 21 in the major cluster of T4 head genes. All of the genes in this region of the T4 genome have overlapping initiation and termination codons with the sequence T-A-A-T-G. We present the DNA sequence of the gene and show that it codes for a protein containing 141 amino acids with an acidic amino-terminal half and a basic carboxyl terminus. Antibodies prepared against the 17K protein were used to show that it is cleaved by the phage-coded gp21 protease during head maturation and that most of the protein leaves the head after cleavage. A frameshift mutation of the gene was constructed in vitro and recombined back into the phage genome. The mutated phages had a drastically reduced burst size and about half of the particles produced were morphologically abnormal, having isometric rather than prolate heads. Thus, the 17K protein is involved in head shape determination but is only semi-essential for T4 growth.     

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides

The pigment content of a B800–850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed.The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800–850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band.The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.     

Conditionally lethal mutants of bacteriophage T4 defective in production of a transfer RNA

The two buried carboxyls (Asp-102 and Asp-194) in both chymotrypsin and chymotrypsinogen are ionized at pH values greater than 4.2 and may be ionized even as low as pH 3.This was demonstrated by coupling most of the surface carboxyis of the proteins by a carbodi-imide with glycinamide or semicarbazide to diminish the groups ionizing at low pH and then titrating the proton uptake on denaturation by sodium dodecyl sulphate between pH 3.0 and 4.6. At pH values greater than 4.2 all unblocked carboxyls are ionized. The proton uptake during the conformational change on denaturation was determined by a stopped-flow procedure and found to be about 2H⁺/mol between pH 3.0 and 3.6. The rate constant for the uptake of protons is the same as that for the exposure of tryptophan and lies in the tens of millisecond region.The buried negative charge at the active site appears to be mainly on Asp-102 rather than on His-57, the pK_a of which must be raised by the buried charge. This enhances its efficacy as a base catalyst in the “charge relay system”.The presence of an intact charge relay system in the inactive zymogen illustrates the importance of stereochemical fit between enzyme and substrate. Enzyme catalysis could hardly be mediated by a catalyst which is uniquely reactive in the absence of correct enzyme-substrate orientation as this would be inconsistent with its specificity.     

Labeling of chromatophore membranes and reaction centers from the photosynthetic bacterium Rhodospirillum rubrum with the hydrophobic marker 5-[125I]iodonaphthyl-1-azide

Chromatophores of the photosynthetic bacterium Rhodospirillum rubrum and isolated reaction centers were labeled with the lipophilic membrane marker 5-[¹²⁵I]iodonaphthyl-1-azide. The two smaller reaction center proteins L and M bind more label than the larger subunit H, a fact supporting the proposed localisation of the 3 subunits obtained with hydrophilic labels. Besides these integral proteins the lipids, among them mainly the pigments and the quinones, are highly labeled suggesting a hydrophobic environment around these molecules and a preferred reactivity to iodonaphthylazide. Such a hydrophobic environment may be of great importance for the function of the photosynthetic reaction centers especially for the charge separation and the primary reactions in electron transport.     

Calmodulin an activator of human erythrocyte (Ca2+ + Mg2+)-ATPase phosphorylation

The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130 000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca²⁺ + Mg²⁺)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca²⁺ + Mg²⁺)-ATPase of human erythrocyte membranes.     

Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and golgi membranes from rat liver

Rough and smooth microsomes and Golgi membranes incorporate $\text{N-}\text{acetylglucosamine}$ from $\text{UDP}\text{-N-}\text{acetylglucosamine}$ into endogenous protein acceptors. A lipid intermediate of the dolichol phosphate type participates in this transfer reaction in the case of both microsomal subfractions, but the nature of lipid glycosylation is different in these two fractions. Glucosamine transfer in Golgi membranes does not appear to involve a lipid intermediate. In contrast to the results obtained under in vivo conditions, no glucosamine label is recovered in nascent ribosomal proteins or on luminal secretory proteins after incubation in vitro. Proteolysis of intact vesicles of the subfractions removes glycosylated dolichol phosphate and protein acceptors to various extents and interferes with transferase activities. This finding suggests the possibility that glycosylation at the cytoplasmic side of the membrane of the endoplasmic reticulum may involve a system separate from that acting at the luminal side of the same membrane.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.